MicroRNAs (miRNAs) serve as novel promising biomarkers for the diagnosis and prognosis of many human diseases. This study investigated the diagnostic value of miR-186-5p for asymptomatic carotid artery stenosis (CAS), and its predictive value for future cerebral ischemic events (CIEs).Sixty-seven cases with asymptomatic CAS and 60 healthy individuals were recruited. Serum levels of miR-186-5p were tested by using qRT-PCR. Receiving–operator characteristic (ROC) curve was drawn based on sensitivity and specificity analyses. All asymptomatic CAS cases were followed up for 5 years. Kaplan-Meier method was applied for the evaluation of the predictive value of miR-186-5p for the occurrence of CIE.The serum level of miR-186-5p was increased significantly in asymptomatic CAS patients. MiR-186-5p was the most significant factor associated with the high degree of carotid stenosis in asymptomatic CAS patients. In the ROC curve analysis, the AUC was 0.919, with the sensitivity of 89.6% and specificity of 81.7% at the cutoff value of 1.221. Kaplan-Meier method results revealed that high miR-186-5p level was associated with the occurrence of CIEs. High miR-186-5p level and high degree of carotid stenosis were independent factors for the occurrence of CIEs.MiR-186-5p serves as a potential diagnostic biomarker for patients with asymptomatic CAS, and predicts the occurrence of future CIEs. Carotid artery stenosis (CAS) is a condition refer to the narrowing of one or both of the carotid arteries. It is usually induced by atherosclerotic lesions, and is considered to be one of the major causes of stroke and cerebral ischemic events (CIE) . ResearcMany studies suggest that a variety of cellular processes such as proliferation, differentiation, and apoptosis can be regulated by microRNAs (miRNAs) , 5. MiRNIn the present study, we analyzed the expression changes of miR-186-5p in the serum of asymptomatic CAS patients by using quantitative reverse-transcription PCR (qRT-PCR). Considering the remarkable changes of serum miR-186-5p in asymptomatic CAS patients, we further investigated its clinical values for the diagnosis and prognosis of asymptomatic CAS.From January 2013 to May 2014, 67 cases diagnosed with asymptomatic CAS were enrolled, and another 60 healthy individuals were recruited as the control group. All study subjects gave written informed consents following the Declaration of Helsinki. The study protocols were approved by the Ethics Committee of Binzhou Medical University Hospital. All cases performed carotid artery color Doppler ultrasonography (CDUS), and the degree of CAS was determined according to the measurement results of computed tomography angiography (CTA). Patients who had conditions that can lead to stroke or CIE were excluded, including atrial fibrillation, myocardial infarction, cardiomyopathy, severe pulmonary disease, and arrhythmia. CAS refers to a > 50% stenosis of the extracranial internal carotid artery (ICA), and the stenosis severity was estimated according to the North American Symptomatic Carotid Endarterectomy Trial method . An asymFasting blood samples were drawn from each individual, and serum samples were collected and stored at − 80 °C after centrifugation. The demographic and clinical information were recorded. Additionally,, all CAS patients were followed-up for 5 years to recorded the occurrence of CIEs, including strokes, transient ischemic attack (TIA) or sudden death. After the CIE occurs, the follow-up was ended.−ΔΔCt method.Firstly, total RNA was extracted from the serum of each subject by using TRIzol reagent  according to the manufacturer’s instructions. Briefly, the serum samples were homogenized in Trizol regnant, then chloroform was added to the 1/5 volume of Trizol and mixed thoroughly. The mixture was centrifuged at 12,000 g (4 °C) for 15 min. The supernatant was collected and mixed with isopropanol at a ratio of 1:1 and incubation for 10 min and was then centrifuged at 14,000 g (4 °C) for 10 min. The supernatant was removed and 100 μl 75% ethanol was added to the tubes and mixed thoroughly. After centrifugation at 8000 g (4 °C) for 5 min, the supernatant was removed and the tubes were air-dried. Then total RNA was reverse transcribed into complementary DNA by using a PrimeScript RT Reagent Kit . Then qRT-PCR was performed on a 7300 Real-Time PCR System  by using a SYBR green I Master Mix Kit . And relative levels of serum miR-186-5p were normalized by U6 by using 2P < 0.05.All statistical analyses were performed by using SPSS 21.0 software  and GraphPad Prism 7.0 software . Categorical variables were presented as counts, and their comparisons between groups were analyzed by using Chi-Square test. Continuous variables were compared between groups by using Student’s t test. Receiving-operator characteristic (ROC) curve was drawn based on sensitivity and specificity analyses. Kaplan-Meier method was applied for the evaluation of the predictive value of miR-186-5p for the occurrence of CIE. The association of different variables with the degree of carotid stenosis was evaluated by using logistic regression analysis. Additionally, the Cox regression analysis was performed to assess independent predictive factors for CIE. The hazard ratios (HRs) or odd ratio (OR) and 95% confidence intervals (CIs) were calculated. Statistical significance was defined as P > 0.05). But DBP (diastolic blood pressure) level was significantly higher in CAS patients group compared with the control group (P < 0.05).As shown in Table P < 0.001).According to the results of qRT-PCR, the serum expression levels of miR-186-5p were compared between healthy individuals and asymptomatic CAS patients. It was found that the serum level of miR-186-5p was increased significantly in asymptomatic CAS patients  and high expression group (n = 36). And the demographic, clinical, and biochemical properties were compared between the two groups. It was found that patients in the high miR-186-5p expression group had high levels of total cholesterol and a high degree of carotid stenosis compared with patients in low miR-186-5p expression group, and a larger proportion of cases suffered from hypertension were found in the high miR-186-5p expression group  of serum levels of miR-186-5p, all asymptomatic CAS patients were divided into low expression group (3 of seruP = 0.025) and total cholesterol levels  were the most significant factors associated with the high degree of carotid stenosis in CAS patients  was 0.919, with the sensitivity of 89.6% and specificity of 81.7% at the cutoff value of 1.221  and high degree of carotid stenosis  were independent factors for the occurrence of CIEs in asymptomatic CAS patients  and conventional transcranial Doppler (TCD) sonography . RecentlIn the present study, we compared the expression level of miR-186-5p in the serum of 67 asymptomatic CAS patients with 60 healthy controls. Analysis of the expression levels of miR-186-5p between two groups showed that miR-186-5p was significantly overexpressed in asymptomatic CAS patients compared with healthy controls, indicating that miR-186-5p might play a crucial role in the occurrence and development of CAS. Consistently, as previous research reported, plasma miR-186-5p shows a remarkable increase in acute coronary syndromes (ACS) patients compared with that in cardiovascular disease (CAD) patients, suggesting the potential role of miR-186-5p in the comprehensive risk stratification of atherosclerotic cardiovascular diseases . These dMiR-186-5p plays a crucial role in a variety of human diseases, and many studies focus on its clinical value for disease diagnosis and prognosis , 22. MiRClinically, analysis of the demographic and clinical data results demonstrated that a larger proportion of cases with high total cholesterol levels or suffer from hypertension were found in the high miR-186-5p expression group, suggesting that higher miR-186-5p level might be associated with dyslipidemia and hypertension in asymptomatic CAS patients. Hypertension is a powerful risk factor for cardiovascular and cerebrovascular diseases, which are important causes of mortality and disability in the elderly . A recenIn summary, the present study highlighted the potential role of serum miR-186-5p for the early diagnosis of asymptomatic CAS. Additionally, overexpression of miR-186-5p was closely associated with the occurrence of CIEs for asymptomatic CAS patients. Collectively, the present results indicated that miR-186-5p serves as a potential diagnostic biomarker for patients with asymptomatic CAS and predicts the occurrence of future CIEs.


N,N’-diethylacrylamide (DEA) and itaconamic acid (IAM) were synthesized by changing the molar ratio of linear copolymer P(DEA-co-IAM) and DEA monomer. Linear copolymer P(DEA-co-IAM) was introduced into a solution of DEA monomer to prepare pH-thermo dual responsive P(DEA-co-IAM)/PDEA semi-IPN hydrogels. The thermal gravimetric analysis (TGA) revealed that the semi-IPN hydrogel has a higher thermal stability than the conventional hydrogel, while the interior morphology by scanning electron microscopy (SEM) showed a porous structure with the pore sizes could be controlled by changing the ratio of linear copolymer in the obtained hydrogels. The oscillatory parallel-plate rheological measurements and compression tests demonstrated a viscoelastic behavior and superior mechanical properties of the semi-IPN hydrogels. Besides, the lower critical solution temperature (LCST) of the linear copolymers increased with the increase of IAM content in the feed, while the semi-IPN hydrogels increased LCSTs with the increase of linear copolymer content introduced. The pH-thermo dual responsive of the hydrogels was investigated using the swelling behavior in various pH and temperature conditions. Finally, the swelling and deswelling rate of the hydrogels were also studied. The results indicated that the pH-thermo dual responsive semi-IPN hydrogels were synthesized successfully and may be a potential material for biomedical, drug delivery or absorption applications. The further applications of semi-IPN hydrogels are being conducted.A series of semi-interpenetrating polymer network (semi-IPN) hydrogels based on  Smart hydrogel material is a three-dimensional network that is able to absorb a large amount of water without dissolving and can undergo a sharp change in volume or phase transition in response to small changes in environmental conditions, such as temperature, pH, ionic strength, light, pressure, redox and electric field ,4,5,6,7.N-substituted polyacrylamides show thermo-responsive behavior, such as poly(N-isopropylacrylamide) (PNIPAM), poly(N-cyclopropyl-acrylamide) (PCPA), poly (PDEA) and poly(N-ethylacrylamide) (PEAM) [In previous reports, the hydrogels of several ) (PEAM) ,13,14. TN-isoprolylacrylamide (NIPAM) was well understood and contributed to their popularity. However, it was reported that during hydrolysis in aqueous solution, PNIPAM-based materials were able to release toxic low molecular weight amines, then their bio-applications were limited [Most of the previous studies about thermo-responsive polymer materials were conducted with PNIPAM which displays a LCST at about 32 °C in pure water . Therefo limited . In this limited  can be a limited . However limited ,20. On t limited . For sev limited ,22 or sy limited ,24, whic limited  into the2 group, has been used as a pH-sensitive segment to prepare thermo-pH dual responsive copolymers [co-IAM) and cross-linker. To the best of our knowledge, this is the first time to combine DEA and IAM together in a semi-IPN hydrogel. FTIR and 1H NMR spectra were used to confirm the chemical structures of the polymers, while the GPC and DLS measurements were used to determine some molecular properties. The other properties were further characterized using TGA, DMA, SEM and rheological measurement. Finally, we combined LCST and swelling behavior measurements in various pH buffer solutions (PBS) and different temperatures to investigate the thermo-pH dual responsive of the obtained semi-IPN hydrogels. It is expected that this novel hydrogel has outstanding performances which being more suited to applications in the life sciences or absorption application. The further applications of obtained semi-IPN hydrogels are being conducted by our research group.In recent years, itaconamic acid (IAM), which has both –COOH and –NHpolymers ,27,28. Ipolymers ,30. In tN,N’-diethylacrylamide  obtained from Tokyo Chemical Industry Co., Ltd. (Japan). Itaconamic acid  was prepared in our laboratory according to the method disclosed in US patent No. 2013/0172490 [4)2S2O8) as an initiator was purchased from Aencore Chemical PTY. Ltd. . N,N,N’,N’-tetramethylethylenediamine  as a catalyst and N,N’-methylenebisacrylamide  as a cross-linker were purchased from Alfa Aesar Co. . N,N-Dimethylformamide (DMF) obtained from Macron Fine Chemicals. Deuterium oxide (D2O) obtained from Sigma Aldrich . The buffer solution of pH 4, pH 7 and pH 10 obtained from J. T. Baker Chemical Corp. , the buffer solution of pH 5 obtained from Alfa Aesar Co. , the buffer solution of pH 6 and pH 8 obtained from Sigma Aldrich . All reactants were used as received without any further purification./0172490 . Ammoniuco-IAM) was prepared by free radical polymerization in aqueous solution of DEA and IAM. APS and TEMED were used as an initiator and accelerator, respectively. The preparation is shown in N,N’-diethylacrylamide , itaconamic acid  and 12.0 mL of deionized (DI) water were mixed together in an one-neck flask by continuous stirring under a nitrogen atmosphere until homogeneous, and placed in an ice-water bath. Then, 2.0 mL aqueous solution of APS 0.084 M and 4.0 mL aqueous solution of TEMED 1.067 M were used to initiate the reaction. The obtained linear copolymer P(DEA-co-IAM) was ready for the subsequent processes after the polymerization in ice-water bath was carried out for 4 h and stabilized about 24 h at room temperature. To purify the obtained copolymer solution for further investigation, about 2.0 mL of it was dialyzed against DI water using a dialysis membrane with molecular weight cut off is 10,000 g/mol for 7 days . Finally, the dried copolymer was received using a vacuum dryer at −50 °C. White solid products were obtained as samples I-1, I-2, I-3, I-4, I-5 for molar ratios of DEA/IAM = 100/1, 100/2, 100/3, 100/4 and 100/5, respectively. For comparison, the linear homopolymer PDEA also was prepared by the same method as described above and named as I-0 sample.Linear copolymer P(DEA-co-IAM) solution and MBA as cross-linker with various designated ratios. APS was used to initiate and TEMED was used to accelerate the reaction. The preparation is shown in co-IAM) I-2 solution, DEA monomer, MBA cross-linker and water were mixed together in ice-water bath for 1 h, under nitrogen atmosphere until homogeneous. After that, initiator and accelerator were added, and the solution was mixed together for 3 min. Then, the solution was quickly poured into a cylindrical glass mold with the diameter of 8 mm, sealed and kept at room temperature for 24 h for the polymerization occurred completely. The obtained hydrogels were removed from the molds, cut into samples with the thickness of 2.5 mm and placed in DI water at room temperature in order to eliminate unreacted materials. The DI water was refreshed several times in about one week. Finally, they were freeze-dried at −50 °C and white freeze-dried hydrogel samples were ready for further investigation.Semi-IPN hydrogels were prepared by free radical polymerization of DEA monomer in the presence of linear copolymer P spectra were recorded on a Bruker Advance 300MHz NMR spectrometer  at room temperature using D2O as the solvent.Fourier transform infrared (FTIR) spectra were carried out by Perkin Elmer Spectrum RXI FTIR instrument  within 4000–400 cmHydrodynamic radii of the linear polymers were confirmed by dynamic light scattering . The 0.5 wt % aqueous solutions of samples were measured at 25 and 45 °C.Mn) and polydispersity index (PDI) of the linear polymers were determined by gel permeation chromatography (GPC) using a Viscotek GPC system from Malvern Ltd.  with DMF as the mobile phase and polystyrene as the standard. About 15 mg of dried sample were dissolved in 5 mL of DMF under following conditions: column 300 mm × 8 mm, flow rate of 1 mL/min, temperature of the column and detector were set to 60 °C, injection quantity of test sample and standard was 150 μL each.The number average molecular weight (co-IAM) copolymers as a function of temperature using a laser transmittance meter (LASOS LGK 7628). The 1.0 wt % aqueous solutions of linear polymer samples were heated from 25 to 50 °C at a heating rate of 1 °C/min. All measurements were taken at a wavelength of 500 nm. The LCST was determined to be the temperature when the transmittance of the solution is 50%.The phase separation profiles and lower critical solution temperatures (LCST) were determined from the transmittance of the sample containing PDEA linear homopolymer or P was used at a heating rate of 1 °C/min in the temperature range of 20–50 °C to confirm the lower critical solution temperature (LCST) of the swelling hydrogels. The temperature at endothermic peak of DSC trace was referred as LCST of the corresponding hydrogel sample.co-IAM), conventional PDEA and semi-IPN hydrogels were tested.A thermal gravimetric analyzer  was used at a heating rate of 10 °C/min in the temperature range of 50–600 °C under a nitrogen atmosphere to investigate thermal stability of the semi-IPN hydrogels. The dried samples of linear copolymer P to investigate the interior morphology. All samples were sputter-coated with gold for 10 min to enhance the conductivity and SEM images were acquired at an accelerating voltage of 15.0 kV. The pore sizes and standard deviations were analyzed by ImageJ.The storage modulus (G’) and loss modulus (G”) were measured to investigate the rheological properties of the swollen hydrogels. Anton Paar MCR 301 rheometer  was used for oscillatory parallel-plate rheological measurements. The G’ and G” were measured as function of time , frequency (in the range of 0.1–10 Hz) and temperature . All tests were performed at a strain of 1%.The stress-strain curves were tested to investigate the mechanical properties of the hydrogels by a dynamic mechanical analyzer  at room temperature. The compression tests were carried out on the cylindrical samples (Φ10 mm × 5 mm) at a rate of 100 mN/min until cracking. The compressive modulus, fracture strain and fracture stress were confirmed from the stress–strain curves. All tests were performed three times.Ws is the weight of the swelling hydrogel and Wd is the weight of the freeze-dried hydrogel.The swelling kinetics of the hydrogels were studied using gravimetric method. The freeze-dried hydrogels were immersed in DI water at 20 °C. At predetermined time points, swelling hydrogels were eliminated the excess water on the surface by filter paper and weighed by an electronic balance. The swelling ratio (SR) was calculated according to the following equation:The thermo-sensitive property of the hydrogels was investigated by calculation the equilibrium-swelling ratio as a function of temperature. The freeze-dried hydrogels were immersed in DI water at different temperature points in the range of 20–50 °C until equilibrium. The swelling hydrogels were eliminated the excess water on the surface by filter paper and weighed by an electronic balance, and SR was calculated using the above equation.The pH-sensitive property of the hydrogels was investigated by calculating the equilibrium-swelling ratio as a function of the pH values. The freeze-dried hydrogels were immersed in pH buffer solution with different pH values in the range of 4–10 at 25 °C until equilibrium. The swelling hydrogels were eliminated the excess water on the surface by filter paper and weighed by an electronic balance, and SR was also confirmed using the above equation.Wt is the weight of the shrunk hydrogel at the specific time t at 37 °C, Wd is the weight of the freeze-dried hydrogel and We is the weight of the equilibrium swollen hydrogel at 25 °C.To investigate the deswelling kinetics of the hydrogels, the freeze-dried samples were firstly immersed in DI water at 25 °C. After one week, the equilibrium swollen hydrogels were quickly transferred to DI water at 37 °C. At predetermined time points, the shrunk hydrogels were removed, wiped off water on the surface by filter paper and weighed by an electronic balance. Water retention (WR) was calculated according to the following equation:co-IAM)/PDEA semi-IPN hydrogels were synthesized by free radical polymerization of DEA monomer in the presence of linear copolymer P(DEA-co-IAM) using MBA, APS and TEMED as cross-linker, initiator and accelerator, respectively. In aqueous solution, the polymerization was initiated by sulfate free radicals, which were produced by the reaction between APS and TEMED [co-IAM) chains were kept and entangled within the hydrogel network through hydrogen bonding interaction.The P. The characteristic peaks from IAM were at δ = 3.3 ppm (–CH2–) and δ = 5.9–6.4 ppm (CH2=). The characteristic peaks from DEA were at δ = 0.9–1.4 ppm ; δ = 3.3–3.5 ppm ; δ = 5.7 (=CH–) and δ = 6.1–6.7 ppm (CH2=). The peaks from DEA at δ = 5.7, 6.1 and 6.7 ppm and from IAM at δ = 5.9 and 6.4 ppm were absent in the spectra of PDEA and P(DEA-co-IAM) while the strong bands at 1.1 and 3.3 ppm correspond to CH3 and CH2 protons of ethyl groups in the side chain; the bands at 1.7 and 2.55 ppm correspond to CH2 and CH protons of the main chain appeared indicating successful synthesis of linear homopolymer PDEA and copolymer P(DEA-co-IAM) [-co-IAM) ,25,33.co-IAM), conventional PDEA and semi-IPN hydrogels are shown in −1, the C–H stretching vibration band of –CH3 and –CH2– groups were shown at about 2924 and 2850 cm−1, and the C=C stretching peak was shown at 1590 cm−1 [−1 and had a distinct characteristic peak at 1710 cm−1 which corresponds to the C=O peak in carboxylic group [co-IAM), conventional PDEA and semi-IPN hydrogels showed an absence of the peak at about 960 and 1590 cm−1, indicating C=C was transformed into C–C. Moreover, the C–H stretching vibration band of –CH3 and –CH2– groups for DEA monomer were also detected for conventional PDEA and semi-IPN hydrogels. In addition, the band at 1710 cm−1 due to the stretching of C=O bond indicates the presence of IAM in the linear copolymer and semi-IPN hydrogel. Thus, the FTIR spectra showed successful synthesis of the linear copolymer P(DEA-co-IAM), conventional PDEA and semi-IPN hydrogels.The FTIR spectra of DEA monomer, IAM comonomer, linear copolymer P(DEA-590 cm−1 . In the co-IAM) solutions at below and above LCST are shown in DH) of around 190.0 and 24.4 nm at 25 and 45 °C, respectively. On the contrary, the polydispersion was observed in the DLS of linear copolymer suggesting the presence of small oligomers and some swollen aggregates of large size . These results can be explained by the influence of IAM monomer. In DI water, the IAM fraction within the linear copolymer P(DEA-co-IAM) could be ionized resulting the charged copolymer chains. Therefore, the aggregation of charged copolymer chains was much easier than that of homopolymer. The same results were noted in the study by Hayde et al. [DH of polymer and copolymer decreased dramatically because the coil-to-globule transition behavior occurred revealing a typical thermally induced aggregation [The temperature-induced transition of linear polymers was investigated by dynamic light scattering (DLS). The DLS results of aqueous linear homopolymer PDEA and linear copolymer P and polydispersity index (PDI) of linear polymers. co-IAM), respectively, while their Mn and PDI are shown in Mn of P(DEA-co-IAM) (5.80 × 104 g/mol) was lower than PDEA (11.5 × 104 g/mol). It may be the influence of methanediyl group (–CH2–) from IAM composition units. In the propagation of free radical polymerization, the unpaired electron at the end of a growing chain could be paired with an electron of carbon-hydrogen bond from methanediyl group of IAM leading to the chain termination occurred quickly. This process is illustrated in the co-IAM) showed one size distribution, while the DLS results presented a polydisperse with three peaks. The main factor explaining this difference could be temperature. As mentioned above, the hydrodynamic radii of linear polymers were measured at 25 and 45 °C, while the molecular weights were confirmed at 60 °C. At high temperatures, the ionization of the IAM segments within linear copolymer P(DEA-co-IAM) took place stronger, so the oligomers could be aggregated together more easily to form the larger globules.Gel permeation chromatography (GPC) was used to investigate the influence of IAM monomer to the number average molecular weight (co-IAM) had a small weight loss of 2% at 170 °C, then the weight loss began to decline rapidly in the range of 260–430 °C and remaining weight was observed about 14% at 600 °C. This result could be explained by the degradation of IAM segments and the rest of these groups linked to DEA, respectively. The same results were reported by Sousa et al. for poly(N-isopropylacrylamide-co-acrylamide) gels [co-IAM) [co-IAM) and PDEA hydrogel proving a strong interaction between the linear copolymer and hydrogel network via hydrogen bonds [N,N-diethylacrylamide-co-methacrylic acid) semi-IPN hydrogel was 13%, which was lower than our current work [co-IAM) possessed higher hydrophilicity than salecan molecules leading to stronger hydrogen bonds in the P(DEA-co-IAM)/PDEA semi-IPN hydrogel. Thermogravimetric analysis (TGA) was carried out to investigate the thermal stability of the hydrogel materials. As shown in de) gels  and Rweien bonds . Moreoveent work . This maco-IAM). Such a phenomenon may be because the hydrophilic groups, such as carboxyl and amino groups in the copolymers, which originated from IAM formed hydrogen bonds with water to increase the LCST. That is, there are more hydrophilic groups in the copolymer than the homo-polymer, and the addition of IAM will significantly increase the LCSTs of obtained copolymers. Besides, the ionization of high hydrophilic units inside the copolymer may interfered in the thermally induced phase separation due to the ionic stabilization [For linear polymers, the phase transition behavior is shown in lization . In thisco-IAM) leading to the increase of the LCST. Similar results were also observed in the previous reports not only for linear copolymers but also for hydrogel materials [For the hydrogels, LCST also is the most important factor demonstrating the thermo-sensitive behavior. In this work, the LCSTs of the hydrogels were measured by DSC analyses and shown in aterials ,27,28,29The photographs of conventional and semi-IPN hydrogel (PDEA2) are described in The SEM images clearly showed the dependence of interior morphology on the linear copolymer content introduced into the semi-IPN hydrogels. As shown in the co-IAM) content introduced into the three-dimensional network. This phenomenon can be explained by the crosslinking density and the flexibility of the resulted hydrogels. More linear copolymer in the semi-IPN materials reduced the crosslinking density and enhanced the flexibility of polymer network due to generation of fewer entanglements in the hydrogel structure, leading to a reduction of elastic modulus [The storage modulus (G’) and loss modulus (G”) of the conventional, as well as semi-IPN hydrogels with different feeding ratios were measured by using an oscillatory rheometer. The  modulus ,45.The influence of temperature on G’ values is shown in co-IAM) within semi-IPN hydrogel acted as a stress-dispersing agent and subjected to higher stress. Similar result was reported by Liu et al. for poly(N-isopropylacrylamide-co-hydroxyethyl methacrylate)/poly(N-isopropylacrylamide) semi-IPN hydrogels [ydrogels . For semydrogels . Figure ydrogels ,30,47,48co-IAM) created a high viscosity medium, which assisted the energy dissipation, took place rapidly during deformation. On the other hand, the semi-IPN hydrogels were high hydrophilic which helped water molecules were kept tighter in the three-dimensional network, leading to increase significantly fracture strain, because water worked as a plasticizer [Another weakness of conventional PDEA hydrogels is low fracture strain compared with that of semi-IPN hydrogels. This can be a significant drawback in reusing a conventional hydrogel for absorption applications. sticizer . The simsticizer ,30,47,48The swelling behavior in DI water at different temperatures was tested to demonstrate the thermo-sensitive property of the obtained hydrogels and the results are shown in On the other hand, when the temperature was increased from 20 to 50 °C , the values of reduction in swelling ratio gradually increased from PDEA0 to PDEA4 samples. For example, PDEA0 had the smallest change in the swelling ratio (ydrogels . This reco-IAM) is a kind of polyampholyte, which contains both anionic and cationic groups originating from IAM units. Thus, the dissociation of carboxylic acid and amino groups was strongly influenced by the pH environment, then significant influenced to swelling behavior. It is clearly seen that the swelling ratios of the hydrogels in pH buffer solutions were much lower than those in DI water because these solutions have a high ionic concentration. As shown in co-IAM) content increased. This can be explained by the high hydrophilic IAM units as discussed above. The swelling behavior in different pH values was tested to demonstrate the pH-responsive properties of the hydrogels. The tendency of swelling ratios in different pH solutions was quite diverse. They were rather high at pH 4, and slightly decreased at pH 5 before reaching the lowest value at pH 7. However, they were raised sharply again at pH 8 and decreased a little at pH 10. This result was also found in semi-IPN hydrogels based on NIPAM and IAM, and was explained by the repulsion of the moieties with the same type of charge in the hydrogel systems . In a neFor many absorption applications, thermo-responsive hydrogel is expected having not only high response rate but also high deswelling rate. co-IAM) and cross-linker MBA. IAM co-monomer reduced the average molecular weight of the linear copolymer, and disappeared at the one-size distribution of the hydrodynamic diameter. Characteristics of the linear copolymer and the hydrogels were investigated by FTIR, 1H NMR, TGA, DSC, SEM, rheological and compressive tests. The chemical structure was confirmed by FTIR and 1H NMR spectra, and suggests that the linear polymer chains were entangled in the three-dimensional network. TGA and mechanical tests revealed the thermal stability and good mechanical properties of the semi-IPN hydrogels. Moreover, the viscoelastic behavior of the obtained hydrogels was also confirmed by rheological tests with the G’ values were much higher than G”. SEM images showed a porous structure of the hydrogels with pore size in the range from 220 ± 77 to 715 ± 83 µm. The LCST of the semi-IPN hydrogels in DI water increased slightly from 31.3 to 33.4 °C as increased the linear copolymer content. The thermo-pH dual responsive of semi-IPN hydrogels could be presented by their swelling behavior in different temperatures and pH buffer solutions: increased linear copolymer content or acid/base solution increased the swelling ratio, and this value dropped rapidly as the temperature increased above LCST. Finally, the swelling and deswelling rates of the semi-IPN hydrogels were also increased quickly with the increase of the linear copolymer content. Thus, the properties of semi-IPN hydrogels absolutely could be controlled by changing the ratio of the linear copolymer introduced into the PDEA network. The phase transition and swelling behavior of the semi-IPN network in this work can be significantly changed by a change of temperature and/or the pH environment, indicating it can be used for a drug delivery system or as a material to absorb heavy metal ions. Further applications of semi-IPN hydrogels based on DEA and IAM are in progress.In this work, a series of thermo-pH dual responsive semi-IPN hydrogels based on DEA and IAM were prepared by the free radical polymerization of DEA aqueous solutions in the presence of the linear copolymer P(DEA-


As a major bioactive compound from grapes, piceatannol (PIC) has been reported to exert anti-atherosclerotic activity in various studies. Nevertheless, the mechanism underlying the effect of piceatannol against atherosclerosis (AS) is elusive. Our study identified miR-200a/Nrf2/GSDMD signaling pathway as critical mediators in the effect of piceatannol on macrophages. In the present study, we confirmed that treatment of piceatannol repressed the oxLDL-induced lipid storage in macrophages. Compared with control group, piceatannol inhibited TG storage and the activity of caspase1. It is noting that in response to oxLDL challenge, piceatannol abated the pyroptosis in RAW264.7 cells, with a decreased expression of caspase1, gasdermin D (GSDMD), IL-18, IL-1β and NLRP3. Moreover, we investigated the role of microRNA (miR)-200a/Nrf2 signaling pathway in the effect of piceatannol. The results declared that after transfection of si-miR-200a or si-Nrf2 plasmids, the effects of piceatannol on macrophages were converted, including lipid storage and pyroptosis. Importantly, si-miR-200a plasmid reduced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), indicating that miR-200a acted as an enhancer of Nrf2 in macrophages. Collectively, our findings demonstrate that piceatannol exerts anti-atherosclerotic activity on RAW264.7 cells by regulating miR-200a/Nrf2/GSDMD signaling. The present study is the first time to identify miR-200a as a candidate target in AS and declared an association between miR-200a and pyroptosis, which provides a novel therapy for the treatment of AS. As a chronic vascular disease, atherosclerosis (AS) is currently seriously affecting human health. AS is associated with multiple risk factors, such as hyperlipidemia, hypertension, diabetes, and smoking . A dysfuAs a natural phenolic compound with similar chemical structure to resveratrol, piceatannol (PIC) is believed to exert beneficial effects similar to resveratrol . It has As one of the most essential transcription factors, nuclear factor erythroid 2-related factor 2 (Nrf2) exerts antioxidant, anti-apoptotic, and anti-inflammatory effects by interacting with multiple signaling pathways ,13. It hIn the present study, we found that piceatannol attenuated the lipid accumulation in macrophages in response to oxLDL challenge, which was associated with decreased level of pyroptosis. It is noting that up-regulation of miR-200a/Nrf2 signaling was also identified in the study. Additionally, we explored the effect of miR-200a/Nrf2 signaling on piceatannol treatment to protect against lipid accumulation in macrophages. Our study may provide new insights into the therapy of AS.2 under a water-saturated atmosphere in a cell incubator at 37°C. The trans-isomer of piceatannol (PIC) was purchased from Sigma–Aldrich, St. Louis, MO, U.S.A. . DMSO  was used as the negative control.RAW264.7 macrophage cells were kindly gifted by Dr. Li from Peking University in China, and cultured in RPMI 1640 medium, which contained 10% FBS  and 1% penicillin–streptomycin . Cells were cultured in 5% CO7 cells/ml for 24 h. Lipofectamine 2000 reagent  was used for transfection according to the manufacturer’s instructions. After transfection for 48 h, cells were collected for further researches.The si-Nrf2 plasmid, miR-200a mimic, and their negative controls were procured from GenePharma . The RAW264.7 cells were cultured in six-well plates at a density of 1 × 106 cells/well) were cultured in 24-well microplates, and then seeded overnight at 37°C in a 5% CO2 incubator. Furthermore, the cells were treated with PIC (30 μM) for 48 h. All protein was extracted by using cell lysis buffer  from the macrophages. After that, centrifugation was used to clear lysates, following with a measurement of protein concentration by BCA protein assay kit . Caspase1 activity kit  was used according to the manufacturer’s instructions. The plate reader was used to determine the absorbance values at 405 nm. The activity levels were expressed compared with the control group.RAW264.7 cells  for 30 min to examine intracellular lipid accumulation. Commercial kits  were used to measure the contents of total cholesterol (TC) and triglyceride (TG) in the macrophages, according to the manufacturer’s instructions.First, cells were centrifuged at 1000 rpm for 2 min. Subsequently, according to the manufacturer’s instructions, relative LDH release was determined using a commercial kit . The absorbance of samples was assessed at 490 nm using a microplate reader.After all treatment, RAW264.7 cells were lysed in RIPA lysis buffer , containing 1% protease inhibitor cocktail  and 1% phosphatase inhibitor . The protein concentration of lysate was determined by using BCA Assay Kit . Twenty micrograms of protein was isolated using 10% SDS/PAGE and electro-transferred to PVDF membranes . The membranes were blocked by TBST containing 5% milk for 2 h at room temperature, and probed with various primary antibodies at 4°C overnight. After being washed with TBST at intervals of 10 min, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After washing for five times, the immunoreactive bands were visualized using Clarity Western ECL Substrate . The protein bands were visualized with a Bio-Rad System . Actin served as a loading control.Antibodies against caspase1 (A16792), actin (AC026) were obtained from ABclonal . Antibodies specific for IL-1β (ab9722), IL-18 (ab191152), GSDMD (ab210070), NLRP3 (ab263899), Nrf2 (ab92946) were purchased from Abcam . The dilution ratio for all primary antibodies was 1:2000. The secondary antibodies used in the present study were peroxidase AffiniPure goat anti-rabbit-IgG (H+L) (BF03008X) and goat anti-mouse-IgG (H+L) (BF03001X) obtained from Biodragon . Secondary antibodies were used at 1:5000 dilution.-actin expression. The primer pairs used in the present study are listed in Supplementary Table S1.Briefly, TRIzol reagent  was used to isolate total RNA from RAW264.7 cells. The extraction of RNA was conducted on the basis of the manufacturer’s instructions, along with the transcription of RNA samples into cDNA employing a Transcriptor First-Strand cDNA Synthesis Kit . The ABI-Quant Studio 5 system and the SYBR Green PCR Master Mix Reagent Kit  were used to quantify the PCR amplification products. The mRNA expression levels of the target genes were normalized to βt test or one-way ANOVA. Data from all studies were collected in a blinded fashion. No data were excluded when performing the final statistical analysis. The difference was statistically significant at P<0.05.Experimental data were expressed by mean ± SEM. Using GraphPad Prism 8.0 software  to analyze differences among the various treatment groups by two-tailed Student’s To determine the effect of PIC A on oxLDFurthermore, we speculated whether PIC treatment affected the level of pyroptosis. Hence, real-time PCR analysis was carried out to detect the level of pyroptosis. As expected, the results showed that PIC inhibited pyroptosis of macrophages when exposed to oxLDL. In mRNA level, GSDMD and caspase1 expression were down-regulated after PIC administration as compared with the cells without PIC treatment A. We alsIt has been well known that Nrf2 was up-regulated in the effect of PIC against AS. Therefore, we postulated that PIC might target Nrf2 to modulate pyroptosis in macrophages. Notably, we found that both in mRNA and protein level, PIC up-regulated Nrf2 expression in macrophages A,B. MorePrevious studies provided a robust evidence to support a cross-talk between Nrf2 and miR-200a . Hence, AS is a chronic cardiometabolic disease, and remains the leading cause of deaths around the world . Due to As a novel form of cell death, pyroptosis is uniquely dependent on caspase1 which is not involved in apoptosis ,26. It hFurthermore, we explored the role of miR-200a/Nrf2 signaling pathway in the effect of PIC on macrophages. As a master regulator of cytoprotective genes, Nrf2 is known for its antioxidant and anti-inflammatory properties . AccordiIt is worth noting that as an upstream of Nrf2, miR-200a has been reported to regulate the Nrf2 activation. Elevated expression of miR-200a inhibits Keap1 and up-regulates Nrf2 nuclear translocation, which results in a decrease in ROS production in human adult cardiomyocyte line in response to hypoxia exposure . It has Collectively, we identified PIC as a potential candidate for the treatment of AS by regulating miR-200a/Nrf2/GSDMD signaling pathway. When exposed to oxLDL, the PIC administration inhibited pyroptosis and lipid accumulation in macrophages by up-regulating miR-200a and Nrf2. Transfection of si-miR-200a or si-Nrf2 plasmids reversed the effect induced by PIC. It is the first time to declare that PIC alleviates AS by inhibiting pyroptosis, and demonstrated an association between miR-200a and pyroptosis. The present study provides evidences to support PIC to be regarded as a strong option for therapy of AS.Click here for additional data file.


Perinatal asphyxia (PA) is responsible for a large proportion of neonatal deaths and numerous neurological sequelae, including visual dysfunction and blindness. In PA, the retina is exposed to ischemia/reoxygenation, which results in nitric oxide (NO) overproduction and neurotoxicity. We hypothesized that methylene blue (MB), a guanylyl cyclase inhibitor, and free-radical scavenger currently used in the clinic, may block this pathway and prevent PA-induced retinal degeneration. Male rat pups were subjected to an experimental model of PA. Four groups were studied: normally delivered (CTL), normally delivered treated with 2 mg Kg-1 MB (MB), exposed to PA for 20 min at 37°C (PA), and exposed to PA and, then, treated with MB (PA-MB). Scotopic electroretinography performed 45 days after birth showed that PA animals had significant defects in the a- and b-waves and oscillatory potentials (OP). The same animals presented a significant increase in the thickness of the inner retina and a large number of TUNEL-positive cells. All these physiological and morphological parameters were significantly prevented by the treatment with MB. Gene expression analysis demonstrated significant increases in iNOS, MMP9, and VEGF in the eyes of PA animals, which were prevented by MB treatment. In conclusion, MB regulates key players of inflammation, matrix remodeling, gliosis, and angiogenesis in the eye and could be used as a treatment to prevent the deleterious visual consequences of PA. Given its safety profile and low cost, MB may be used clinically in places where alternative treatments may be unavailable. Perinatal asphyxia (PA) is the most severe perinatological problem across the world  and its target genes such as vascular endothelial growth factor (VEGF), adrenomedullin (AM), and inducible nitric oxide synthase (iNOS), among others  or a dose of 2 mg Kg−1 methylene blue in saline solution . The same procedure was implemented with asphyctic newborns to generate the other two experimental groups: asphyctic animals that received saline  or methylene blue treatment .Severe PA was induced using a noninvasive model of hypoxia-ischemia as described  were subjected to scotopic electroretinography, as described  + 5 mg/Kg xylazine  under dim red illumination. An ophthalmic solution of 5% phenylephrine hydrochloride and 0.5% tropicamide  was used to dilate the pupils. Rats were placed facing the stimulus at a distance of 25 cm in a highly reflective environment. A reference electrode was placed through the ear, a grounding electrode was attached to the tail, and a gold electrode was placed in contact with the central cornea. Scotopic electroretinograms (ERG) were recorded from both eyes simultaneously and 20 responses were collected to flashes of unattenuated white light  from a photic stimulator (light-emitting diodes) set at maximum brightness. The registered response was amplified (9 cd s/m2 without filter), filtered , and averaged . The a-wave was measured as the difference in amplitude between the recording at onset and the trough of the negative deflection and the b-wave amplitude was measured from the trough of the a-wave to the peak of the b-wave. Values from each eye were averaged, and the resultant mean value was used to compute the group’s mean a- and b-wave amplitudes ± SEM. To calculate oscillatory potentials (OP), the same photic stimulator was used with filters of high (300 Hz) and low (100 Hz) frequency. The amplitudes of the OP were estimated by using the peak-to-trough method. The sum of three OP was used for statistical analysis.Forty-five days after birth, young rats . Animals were decapitated. After enucleating, anterior segments of the eyes, including the lens, were discarded, and the posterior segments of the eyes containing the retinas were fixed in 4% paraformaldehyde in 0.1 M pH 7.4 phosphate buffer at 4°C for 48 h. Tissues were dehydrated and paraffin-embedded. Tissue sections (5 μm-thick) were stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) with the in situ Cell Death Detection POD Kit , following manufacturer’s instructions. Visualization of immunoreactivity was performed with 0.03% 3,3′diaminobenzidine , 3% nickel ammonium sulphate and 0.01% hydrogen peroxide diluted in 0.1 M buffer acetate, yielding a black product.Rats on the four experimental groups were sacrificed 6 days postpartum , which includes the internal limiting membrane, the retinal optic nerve fiber layer, and the ganglion cell layer (GCL), as reported . Non-specific binding was blocked by exposure to 10% normal donkey serum  for 30 min, and then tissue sections were incubated with primary antibodies , overnigThe eyes of four animals of each experimental group were analyzed. Care was taken on selecting anatomically matched areas of retina among animals before assays. The central area of the sagittal plane was chosen for each retina. The thickness of all layers in the retina was measured in 10 fields using Scion Image software. Special attention was paid to the inner retina (IR), which includes the internal limiting membrane, the optic retinal nerve fiber layer, and the GCL.TUNEL-positive cells were counted on the retinas of four eyes per experimental group. Fields were chosen in the central region of retinal cross-sections and the number of positive cells in ten fields, 500 μm in length, was recorded.n = 4 per experimental group) were sacrificed at different times after MB treatment . The posterior chambers of the eyes were homogenized with TRIzol  and RNA was isolated with RNeasy Mini kit including a DNAse I on-column digestion . One microgram of total RNA was reverse-transcribed into first-strand cDNA using random primers and the SuperScript III kit (Invitrogen) in a total volume of 20 μl according to the manufacturer’s instructions. Reverse transcriptase was omitted in control reactions, where the absence of PCR-amplified DNA confirmed the lack of contamination from genomic DNA. Resulting cDNA was mixed with SYBR Green PCR Master Mix  for quantitative real-time polymerase chain reaction (qRT-PCR) using 0.3 μM forward and reverse oligonucleotide primers  reduction of the a- and b-wave amplitude (p < 0.05) in the amplitude of both waves (p < 0.05) and b- (p < 0.001) waves more similar to the controls than to the PA animals  in the OP patterns (p < 0.001) restored the control pattern (Similar observations were made when studying the OP . PA-indupatterns  whereas  pattern .p < 0.0001) thicker IR (p < 0.0001) prevented this morphological manifestation of the pathology (p < 0.0001) compared to the control groups. In the PA-MB group, the number of ganglion cells was significantly higher (p < 0.0001) than in the PA group , and an increased number of GFAP-positive cellular processes  increase in the number of apoptotic cells (arrows) in the GCL (p < 0.0001) reduced the number of apoptotic cells in the retina of asphyctic animals  increase of iNOS in the PA animals when compared to CTL and MB groups. Treatment of PA animals with MB completely prevented iNOS overexpression (p < 0.05). Other inflammation markers whose expression was modulated by MB included IL1β at 12 h (p < 0.05) decrease in the PA-MB group when compared to the PA animals.Gene expression was studied in the retina for several markers  at diffe at 12 h  and TNFα at 12 h . In bothp < 0.05) increased on the PA group at 12 h and this pathological increase was fully prevented (p < 0.05) by MB treatment  are key enzymes on tissue remodeling. We did not see any significant modulation of MMP2 at any time by either asphyxia or MB treatment . On the reatment . Coincidreatment .p < 0.05) increase of VEGF expression in the retinas of asphyctic rats, and this increase was significantly counteracted (p < 0.05) by MB treatment (p < 0.001) increase in the PA-MB group when compared to all other experimental groups , and the b-wave by the ganglion cells .JF, RP, MR-F, MS, DC, VD, and JL-C: acquisition, analysis, and interpretation of data. IML, CFL, and AM: conception and design, analysis and interpretation of data. AM: wrote the article. All authors revised the original manuscript and agreed on its contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An inverted-type InAlAs/InAs metal-oxide-semiconductor high-electron-mobility transistor (MOS-HEMT) with liquid phase oxidized (LPO) InAlAs as the gate insulator is demonstrated. A thin InAs layer is inserted in the sub-channel layers of InGaAs to enhance the device performance. The proposed inverted-type InAlAs/InAs MOS-HEMT exhibits an improved maximum drain current density, higher transconductance, lower leakage current density, suppressed noise figures, and enhanced associated gain compared to the conventional Schottky-gate HEMT. Employing LPO to generate MOS structure improves the surface states and enhances the energy barrier. These results reveal that the proposed inverted-type InAlAs/InAs MOS-HEMT can provide an alternative option for device applications. To achieve low noise figures in future radio frequency (RF) applications, InP-based high-electron-mobility transistors (HEMTs) with InGaAs channels have demonstrated very high levels of performance and have been widely used in circuits operating in the microwave range [2/Vs) compared to 53% indium at room temperature. Matsumura et al. [2/Vs, and will thus exhibit high-speed operation and low power consumption. Some studies have also fabricated InAs or InGaAs HEMTs on Si substrates and have shown excellent results [The conventional Si-based field effect transistors are reaching the scaling limit in logic devices . High-inve range . Many efve range  reporteda et al.  reported results ,8. In ad results ,10,11 is0.52Al0.48As Schottky layer with high aluminum content may suffer from the leakage current issue, which causes a reduction in the breakdown voltage. Additionally, an HEMT with high indium content suffers from impact ionization due to the narrow bandgap channel, which induces a higher gate leakage current. One commonly used method to address this issue is to insert a dielectric layer between the metal gate and the In0.52Al0.48As layer to supply a high energy barrier. Several laboratories have announced successful results related to the preparation of oxide film on semiconductors, such as thermal oxidation [An Inxidation , chemicaxidation , photochxidation , moleculxidation , and atoxidation . In partxidation  and wet xidation  has beenxidation  operatedxidation ,21,22 an4NO3) and a little nitric acid. When the growth solution was heated using a temperature-controlled heater , the hydrolysis reaction of the ammonium nitrate solution generated ammonia gas and nitric acid. This can be described as Formula (1). Nitric acid oxidizes GaAs-based materials because it is a strong oxidizing reagent. In general, nitric acid is easier to decompose due to absorption heat and can be described as Formula (2).For preparing the LPO growth solution, gallium-ion-containing nitric acid solution was obtained through the sufficient dissolution of high purity (6N) gallium metal in heat (60 °C) and concentrated nitric acid (70%) for more than 8 h, and was then diluted with deionized (DI) water . Next, t2O3, As2O3, and In2O3. However, there were almost no Al oxides on the surface after 1 h oxidation, which was similar to the results in reference [eference . Figure 18 cm−3, a 6 nm undoped InAlAs spacer layer, a 3 nm undoped In0.53Ga0.47As sub-channel layer, a 2 nm undoped InAs channel layer, a 13 nm undoped In0.53Ga0.47As sub-channel layer, a 20 nm undoped InAlAs Schottky layer, and a 2.5 nm undoped InAs capping layer. Room temperature Hall measurements showed electron mobility of 14,262 cm2/Vs and a sheet carrier concentration of 2.07 × 1012 cm−2. 3PO4-based solution. Mesa etching should reach the buffer layer. After forming the ohmic alloy with Au/Ge/Ni by evaporation and annealing at 420 °C for 30 s in an N2 atmosphere, gate recess was performed using a citric-acid-based etchant. For MOS-HEMT fabrication, LPO was used to generate the gate insulator by immersing the post-etching samples into the growth solution. The thickness of gate oxide was approximately 11 nm. Finally, the gate metal was deposited with Au for the reference HEMT and the MOS-HEMT. The device structure was grown using the MBE system on a 3 inch semi-insulating InP substrate. It was composed of a 300 nm undoped InAlAs buffer layer, a 6 nm InAlAs donor layer with a silicon doping density of 4 × 10DS) versus the drain-to-source voltage (VDS) characteristics of the conventional HEMT and MOS-HEMT with a gate length and width of 1 μm and 100 μm, respectively. The gate-to-source voltage (VGS) ranged from −1.4 to 1.4 V in 0.4 V steps. The maximum IDS of the reference HEMT was 441 mA/mm at VDS = 1.5 V. The maximum IDS of the MOS-HEMT was 509 mA/mm at VDS = 1.5 V, which is higher than that of the reference HEMT. It was difficult to obtain the completed pinch-off characteristics for both devices. The reason why such devices do not turn off completely has been attributed to the carrier supply layer being far away from the metal gate or donor-type traps located in the lower portion of the energy bandgap at the InAlAs/oxide interface [nterface , which mDS and related transconductance (gm) versus VGS at VDS = 1.5 V for both devices are shown in m was 243 mS/mm and the threshold voltage was −1.9 V for the reference HEMT. The maximum extrinsic gm was 327 mS/mm and the threshold voltage was −1.7 V for the proposed MOS-HEMT. The band bending in the InAlAs Schottky layer is affected at the higher gate bias, and the conduction band has a chance to be lower than the Fermi level, leading to a small gm at higher VGS. The enhanced IDS and gm characteristics were attributed to the improvement in the oxide/InAlAs interface.The IGS for the reference HEMT and the proposed MOS-HEMT, respectively. The VDS ranged from 0.5 to 1.5 V in 0.5 V steps. The gate current density for the MOS-HEMT was significantly suppressed by at least two orders of magnitude compared to that of the reference HEMT. Please note that this is expressed on a logarithm scale, so the change in the current density is obvious. The enhanced impact ionization was due to the higher electric field existing around the gate-to-drain region where the hot carrier phenomena occurred in the narrow composite channel in the case of the reference HEMT. In the MOS-HEMT, the electric field was weaker than that of the Schottky-gate HEMT owing to the oxidized InAlAs between the metal gate and the InAlAs Schottky layer.GD) for both devices, respectively. In this measurement, the source terminal was floating and the drain terminal was grounded. The turn-on voltages of the Schottky-gate HEMT and the MOS-HEMT were 1.54 and 3.98 V, respectively. The corresponding reverse gate-to-drain breakdown voltages of the reference HEMT and the MOS-HEMT were −2.5 and −4.6 V, respectively. In general, a higher turn-on voltage or reverse gate-to-drain breakdown voltage accompanies a smaller gate leakage current and higher gate voltage swing. In addition, a larger turn-on voltage allows a higher IDS in the composite channel, which improves the device power and is consistent with the results shown in min) can be obtained as Formula (6):f is the operating frequency; fT is cutoff frequency; Kf is the Fukui factor; Rs is the source series resistance; Rg is the gate series resistance [With the increasing demands of wireless communication, applications with low noise figures and high associated gains are required ,25. Circsistance . The forThe main noise sources of HEMTs include shot noise, hot-electron noise, and generation-recombination noise ,28. The m compared with those of the reference HEMT. Besides, a suppressed noise figure and enhanced associated gain can be achieved based on the improved gate current density, decreased impact ionization, and reduced interface traps due to the oxidized InAlAs film. These results indicate that the proposed inverted-type InAlAs/InAs MOS-HEMT can provide an alternative option for device applications.In this study, we used the LPO to grow oxidized InAlAs film on a Schottky layer for fabricating inverted-type InAlAs/InAs MOS-HEMT. A thin InAs layer was inserted within the InGaAs sub-channel layer to enhance the device performance. The proposed inverted-type InAlAs/InAs MOS-HEMT exhibited higher drain current density and g


Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), an aggressive form of T-cell malignancy. The relationship between HTLV-1 infection and cancer progression is controversial. HTLV-1 encodes oncogenic protein TAX1 and it is hypothesized that HTLV-1 infection is associated with breast cancer progression. In this study, we evaluated the relationship between HTLV-1 infection and clinicopathological factors in breast cancer patients. We retrospectively analyzed 610 patients with primary breast cancer who underwent surgical treatment without preoperative chemotherapy at Kagoshima University Hospital between January 2001 and January 2015. When patients with and without HTLV-1 infection were compared, no differences in clinicopathological factors were observed, except for age. Disease-free survival and overall survival rates did not differ between groups. HTLV-1–positive patients were significantly older than HTLV-1–negative patients. It was supposed to be due to the fact that the HTLV-1 infection rate is decreasing. Any effect of HTLV-1 infection on breast cancer progression appears to be negligibly small. Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes an aggressive form of T-cell malignancy called adult T-cell leukemia/lymphoma (ATL) . The association between HTLV-1 infection and cancer occurrence is controversial. HTLV-1–infected patients are reported to be at significantly high risk of developing liver cancer and lymphomas other than ATL . However, a lower incidence of HTLV-1 in gastric cancer patients compared to the general population suggests that HTLV-1 infection reduces the risk of gastric cancer . To the best of our knowledge, no reports have demonstrated a significant difference in breast cancer incidence between HTLV-1–infected patients and uninfected patients. Tax1, a protein encoded by HTLV-1, has demonstrated oncogenic properties via the Ras-Raf-MEK-ERK signaling pathway in breast cancer cell lines . Thus, HTLV-1 infection has been hypothesized to be associated with breast cancer progression. In this study, we evaluated the relationship between HTLV-1 infection and clinicopathological factors in breast cancer patients.PatientsWe enrolled 686 patients with primary breast cancer who underwent curative surgical treatment without preoperative chemotherapy at Kagoshima University Hospital, Kagoshima, Japan between January 2001 and January 2015. During this period, HTLV-1 infection status was checked as part of the preoperative routine examination. Diagnostic method for HTLV-1 infection was chemiluminescence immunoassay against HTLV-1 antigen. Patients with recurrent breast cancer and those for whom HTLV-1 infection status was not checked were excluded. As a result, 610 patients were included in the present analysis. Clinicopathological information was collected retrospectively using medical records, and the seventh edition of the TNM classification was used for staging. Adjuvant therapy was selected for each patient based on the National Comprehensive Cancer Network guidelines. Statistical analysisDifferences in clinicopathological factors between groups were evaluated using analysis of variance for continuous variables and the Pearson chi-squared test for categorical variables. Disease-free survival (DFS) and overall survival (OS) were measured from the time of first surgery until the date of death or last follow-up. Survival curves were calculated using the Kaplan-Meier method, and statistical significance between groups was assessed using the log-rank test. Statistical analysis was performed using JMP Pro, version 12.1.0 for Mac OS . P values <0.05 were considered statistically significant.Patient characteristics Clinicopathological characteristics of enrolled patients are shown in Table 1. The mean age was 60.4 years, and 11% (n=66) of patients were HTLV-1–positive. No patient had ATL within the observation period. Immunohistochemical analysis was not performed in a small number of patients. Additionally, HER2 and Ki67 expression were not evaluated in some patients with ducal carcinoma in situ. The recurrence rate was 14% (n=85) among patients with stage 0-III disease, and 14% of patients (n=85) died during the observation period.Comparison of clinicopathological characteristics between patients with and without HTLV-1 infectionWhen patients with and without HTLV-1 infection were compared, no differences in clinicopathological factors were observed, except for age (Table 2); HTLV-1–positive patients were significantly older than HTLV-1–negative patients. Rates of recurrence and death were not different (Table 2), and DFS and OS did not differ between groups .HTLV-1 is a retrovirus that causes ATL, an aggressive form of T-cell malignancy . HTLV-1 is transmitted from mother to infant via breast milk and is known to be prevalent in several specific areas in the world, including Southwestern Japan . It was estimated that there were 10-20 millions HTLV-1 infected individuals in the world . In Japan, HTLV-1 infection has been extensively studied for many years, and infected individuals was estimated to be around 1.2 million . The geographic distribution of HTLV-1 carriers is quite uneven in Japan and the greatest prevalence is observed in Southwestern Japan . Our hospital located at Kagoshima in Southwestern Japan, and Kagoshima was reported to have highest HTLV-1 seropositive women rate in the world . So, we routinely checked HTLV-1 infection status for all preoperative patients for a period of time. In the present cohort, 11% of patients had HTLV-1 infection. Although this infection rate is relatively high, it is compatible with previously reported rates in our geographic region. To the best of our knowledge, no reports of a significant association between HTLV-1 infection and breast cancer occurrence have been published. We did not have access to data from healthy controls to compare with the patients from the present cohort, so we could not analyze the association between HTLV-1 infection and breast cancer incidence. Rather, in this study we aimed to evaluate the effect of HTLV-1 infection on clinicopathological factors following breast cancer diagnosis. No significant differences were observed between HTLV-1–positive and –negative patients, except for age: the age at diagnosis was significantly higher among HTLV-1–positive patients compared HTLV-1–negative patients. This may be due in part to the fact that the HTLV-1 infection rate is decreasing. Overall, Satake et al. (2012) reported that the number of HTLV-1 carriers has decreased 10% over the past 2 decades (1980s to 2000s), so the age distribution of carriers has trended toward older patients .HTLV-1 encodes Tax1, which was originally identified as a transcriptional activator that plays a central role in the immortalization of infected T-cells . Tax1 has been reported to promote cancer cell proliferation via the Ras-Raf-MEK-ERK signaling pathway . In addition, Tax1 sensitizes breast cancer cells to malignant transformation induced by environmental carcinogens through the inhibition of estrogen-induced ERα-mediated BRCA1 expression . Thus, we hypothesized that HTLV-1 infection is associated with breast cancer progression. However, no significant differences in tumor size, lymph node metastasis, stage, or other factors were observed between HTLV-1–positive and –negative patients. Furthermore, with respect to prognosis, no significant differences in recurrence or OS were observed. Any effect of HTLV-1 infection on breast cancer progression therefore appears to be negligibly small.This study was approved by the institutional review board of Kagoshima University Hospital. And for this type of study formal consent is not required.There is no financial relationships or other interests associated with this manuscript, which might be construed as constituting a conflict of interest.


Background: The CheckMate 227 trial has indicated that nivolumab plus ipilimumab compared with chemotherapy significantly increases long-term survival in the first-line setting of advanced non-small-cell lung cancer (NSCLC).Methods: A Markov model was built to estimate the cost and effectiveness of nivolumab plus ipilimumab vs. chemotherapy as the first-line therapy in patients with advanced NSCLC based on outcomes data from the CheckMate 227 trial. We calculated the cost and health outcomes at a willingness-to-pay (WTP) threshold of $150,000 per quality adjusted life year (QALY) in populations with different programmed death ligand 1 (PD-L1) expression levels  or a high tumor mutational burden (TMB) (≥10 mutations per megabase). Sensitivity analysis were used to test the model stability.Results: The outcomes showed that the incremental costs and QALYs by using nivolumab plus ipilimumab were $124180.76 and 1.16, $70951.42 and 0.53, $144093.63 and 0.83 for the advanced NSCLC patients with a PD-L1 expression ≥50%, ≥1%, and <1%, which led to an incremental cost-effective ratio (ICER) of $107403.72, $133732.20, and $172589.15 per QALY, respectively. For patients with a high TMB, nivolumab plus ipilimumab contributed an extra 2.04 QALYs at a cost of $69182.50 per QALY.Conclusion: Nivolumab plus ipilimumab as first-line therapy makes a better cost-effective strategy than chemotherapy in advanced NSCLC patients with PD-L1 expression levels ≥50% and ≥1% or a high TMB, at a willingness-to-pay threshold of $150,000 per QALY, but not in the patients with a PD-L1 expression <1%. Around the globe, lung cancer is the leading cause of cancer incidence and mortality, with 2.1 million new lung cancer cases and 1.8 million deaths worldwide . Up to 6Nivolumab, the fully human immunoglobulin G4 monoclonal antibody inhibitor of programmed death-1 (PD-1), and ipilimumab, a fully human immunoglobulin G1 monoclonal antibody that targets the cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint receptor, are immune checkpoint inhibitors with distinct but complementary mechanisms of action. In preclinical and clinical settings, the combination of nivolumab plus ipilimumab has presented enhanced activity over nivolumab monotherapy's, which has been approved for the treatment of metastatic melanoma and renal-cell carcinoma –16. In tTo our knowledge, it is still unclear whether the use of first-line nivolumab and ipilimumab would be cost-effective for advanced NSCLC with different PD-L1 expression levels. This study aims to evaluate the cost-effectiveness of nivolumab plus ipilimumab vs. chemotherapy in previous untreated advanced NSCLC patients without driver alterations that can be targeted. The cost-effectiveness analyses were conducted, respectively, in three populations with different PD-L1 expression levels  or patients with a high tumor mutational burden (TMB) (≥10 mutations per megabase), using the most recently reported data from CheckMate 227 –19.A Markov model was constructed on the basis of outcomes data from the CheckMate 227 trial to evaluate the costs and effectiveness of using nivolumab plus ipilimumab vs. chemotherapy as first-line therapy for advanced NSCLC from the US payer's perspective. The Markov model cycle length was 6-weeks and the time horizon were 20-years. We adopted a 3% discount rate per year for both costs and outcomes , progressive disease (PD), and death . Patienthttp://www.getdata-graph-digitizer.com/index.php) was applied to extracting the data points from the OS Kaplan-Meier curves reported in the CheckMate 227 trial, and these data points were then used to fit parametric survival models. The Weibull survival curves matched the number of patients in three states including PFS, PD and death overtime, as the Weibull distribution was flexible and widely used in cancer survival analysis according to Akaike information criterion. Then, we estimated the shape parameter (γ) and the scale parameter (λ) from this fit, and applied Kaplan-Meier curves by using R software package (http://www.r-project.org) and the method of Hoyle et al.  = exp(−λtγ) .while S ∧Shape-Scale*(_stage+1) ∧Shape)1-exp  and those with a high TMB (≥10 mutations per megabase).Utility was adopted to measure patient's preference for living at a particular health state that is often referred to as QALYs (0 stood for death and 1 for perfect heath), which reflected the impacts of the disease-related health states. We used utilities of 0.784 and 0.693 for the patients with nivolumab plus ipilimumab and chemotherapy as the first-line therapy, respectively, based on the patient-reported outcomes results from CheckMate 227 trial . The pre2 of body surface area) plus cisplatin (75 mg/m2) or carboplatin , and for squamous NSCLC, with gemcitabine  plus cisplatin (75 mg/m2) or gemcitabine  plus carboplatin }(2 every 3 weeks)  plus ipilimumab (1 mg per kilogram every 6 weeks). The chemotherapy group were treated with platinum-doublet chemotherapy every 3 weeks for up to four cycles{non-squamous NSCLC were treated with pemetrexed }, 18. Aft3 weeks) .2, and serum creatinine, 1  in the field of lung cancer. Previous studies suggested that combination immunotherapy with nivolumab plus ipilimumab could be considered a cost-effect choice in intermediate- and poor-risk patients with metastatic renal cell carcinoma in the US , 34. HowThe current study is the first cost-effectiveness analysis of nivolumab plus ipilimumab vs. chemotherapy in previously untreated advanced NSCLC patients with different PD-L1 expressions  and a high TMB. Case-based results indicated that the ICERs of nivolumab plus ipilimumab vs. chemotherapy were $107403.72, $133732.20, and $172589.15 per additional QALY in PD-L1 ≥50, ≥1, and <1% populations, respectively. The one-way sensitivity analyses revealed that the cost of nivolumab plus ipilimumab and the utility value of PD were the greatest influence factors in all PD-L1 populations. The probabilistic sensitivity analyses depicted a high likelihood that nivolumab plus ipilimumab would be considered a cost-effective choice at a WTP threshold of $150,000 per QALY in the PD-L1 ≥50 and 1% populations, whereas it is not cost-effective in the PD-L1 <1% populations. Further analysis indicated that the nivolumab plus ipilimumab strategy would be cost-effective at a WTP threshold of $150,000 per QALY for PD-L1 <1% populations by reducing the cost of nivolumab or ipilimumab. In addition, irrespective of tumor PD-L1 expression levels, the ICER of nivolumab plus ipilimumab vs. chemotherapy was $69182.50 for high TMB populations. On the other hand, the results of subgroup analysis exhibited that the ICER of nivolumab plus ipilimumab could be improved by selecting patients in accordance with clinical and pathological parameters , 2.post hoc analysis from CheckMate 026, nonetheless, implied that the application of TMB as a predictive biomarker instead of or in addition to PD-L1 expression may be conducive to selecting patients with advanced NSCLC who embrace great possibility of reaping benefits of immunotherapy  when compared to that in the PD-L1 ≥ 50%  and ≥1%  populations. And further analysis has found that the populations with PD-L1 ≥ 1% did not exclude the PD-L1 ≥ 50% populations, and that the majority of benefits in the former were manifested in the latter. Thus, it may overestimate the cost-effectiveness of benefits of nivolumab plus ipilimumab in patients with PD-L1 expression of 1–49%. Careful deliberation is called for should these patients be to use combination immunotherapy. Furthermore, patients with PD-L1 expression of 1–49% were not estimated in our analysis, because the trial of CheckMate 227 did not report enough survival data on these patients.Like any other models, there are some limitations in our analysis. First, the survival data shows a dramatic tail on the OS curve in CheckMate 227, and the benefits in the dual checkpoint inhibition group will become more significant when compared to those of the chemotherapy with a long-term follow-up. It may underestimate the benefits of combined immunotherapy because our study is based on data of a 29.3-months follow-up. Moreover, our model adds too much weight to PFS and PD. However, most cost-effectiveness analyses of immune checkpoint inhibitors were based on Markov model , 46, 51.When compared to chemotherapy, nivolumab plus ipilimumab as first-line treatment is cost-effective in advanced NSCLC patients with PD-L1 ≥ 50%, PD-L1 ≥ 1% or a high TMB but is not cost-effective in PD-L1 <1% population in view of US payers.The original contributions presented in the study are included in the article/The current research was underlain by model techniques and literature reviews; no written consent was required as per the ethics committee of the Xiangya Hospital of Central South University .HH, LS, and JH conceived and designed the experiments. HH, LS, LY, and DD performed the experiments. HH, LS, LY, DD, JH, YS, and ML analyzed the data. SZ, JH, and DC contributed reagents, materials, and analysis tools. HH, LS, and JH wrote the manuscript. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P = .006), 0.79  and 0.82 , respectively, using the Nyul, WhiteStripe and Z-Score normalization methods compared to no normalization. The relative discretization makes unnecessary the use of intensity normalization for the second-order radiomics features. Even if the bin number for the discretization had a small impact on classification performances, a good compromise was obtained using the 32 bins considering both T1w-gd and T2w-flair sequences. No significant improvements in classification performances were observed using feature selection. A standardized pre-processing pipeline is proposed for the use of radiomics in MRI of brain tumours. For models based on first- and second-order features, we recommend normalizing images with the Z-Score method and adopting an absolute discretization approach. For second-order feature-based signatures, relative discretization can be used without prior normalization. In both cases, 32 bins for discretization are recommended. This study may pave the way for the multicentric development and validation of MR-based radiomics biomarkers.Radiomics relies on the extraction of a wide variety of quantitative image-based features to provide decision support. Magnetic resonance imaging (MRI) contributes to the personalization of patient care but suffers from being highly dependent on acquisition and reconstruction parameters. Today, there are no guidelines regarding the optimal pre-processing of MR images in the context of radiomics, which is crucial for the generalization of published image-based signatures. This study aims to assess the impact of three different intensity normalization methods  typically used in MRI together with two methods for intensity discretization (fixed bin size and fixed bin number). The impact of these methods was evaluated on first- and second-order radiomics features extracted from brain MRI, establishing a unified methodology for future radiomics studies. Two independent MRI datasets were used. The first one (DATASET1) included 20 institutional patients with WHO grade II and III gliomas who underwent post-contrast 3D axial T1-weighted (T1w-gd) and axial T2-weighted fluid attenuation inversion recovery (T2w-flair) sequences on two different MR devices (1.5 T and 3.0 T) with a 1-month delay. Jensen–Shannon divergence was used to compare pairs of intensity histograms before and after normalization. The stability of first-order and second-order features across the two acquisitions was analysed using the concordance correlation coefficient and the intra-class correlation coefficient. The second dataset (DATASET2) was extracted from the public TCIA database and included 108 patients with WHO grade II and III gliomas and 135 patients with WHO grade IV glioblastomas. The impact of normalization and discretization methods was evaluated based on a tumour grade classification task  using five well-established machine learning algorithms. Intensity normalization highly improved the robustness of first-order features and the performances of subsequent classification models. For the T1w-gd sequence, the mean balanced accuracy for tumour grade classification was increased from 0.67 (95% CI 0.61–0.73) to 0.82 (95% CI 0.79–0.84,  Along with machine learning techniques, radiomics is becoming an increasingly popular computer-aided diagnostic tool in the field of medical research3. Radiomics offers an almost unlimited supply of imaging biomarkers that can facilitate cancer detection, diagnosis, and prognosis assessment and the prediction of treatment response4.Radiomics relies on the extraction of a wide variety of quantitative image-based features, including shape, histogram-based, textural and higher order statistics5. Consequently, a large variability in image intensities among inter-patient and intra-patient acquisitions exists that could highly affect the extraction of the radiomics features, compromising the pooling and the reproducibility of published data using independent imaging sets7.Magnetic resonance imaging (MRI) exhibits high soft tissue contrast and submillimetre spatial resolution. In the context of radiomics, a main issue is that MRI intensities are non-standardized and are highly dependent on the manufacturer, sequence type and acquisition parameters10. The variability generated by different voxel sizes can also be reduced by spatial resampling12. Moreover, brain extraction is mandatory to remove the skull regions that generate the most important variations in intensities and to define the region in which intensities should be considered before any image intensity normalization14. However, even though these three types of pre-processing of brain MRI are widely accepted by the community, there is no consensus within radiomics studies regarding the applied image normalization method . The Z-Score method consists of subtracting the mean intensity of the entire image or a region of interest from each voxel value and dividing it by the corresponding standard deviation34.The technique developed by Nyúl et al35. This is an additional critical pre-processing step that does not express any consensus in the literature, and it is usually not truly clarified in radiomics studies  related to the intensity normalization process for both T1w-gd and T2w-flair sequences (DATASET1). On post hoc analysis, significantly higher JSD values were found when comparing images without normalization to Nyul (P < 0.001), WhiteStripe (P < 0.001) and Z-Score (P < 0.001) pre-processed images (Table Jensen–Shannon divergence (JSD) values showed significant differences (P = 0.006), 0.79  and 0.82  when applying the Nyul, WhiteStripe and Z-Score pre-processing methods, respectively (DATASET2) , 0.57 , 0.60  when the Nyul, WhiteStripe and Z-Score methods were applied, respectively  was equal to 0.67  0.61–0.73) when no normalization was applied. In comparison, this value was equal to 0.82 . Regarding the number of bins, only the comparison between 32 and 512 bins demonstrated statistical significance (P = 0.039). For the T2w-flair sequence, the best classification performance was obtained using Nyul’s histogram harmonization and 32 bins, with a mean balanced accuracy of 0.67  as opposed to the impact of discretization (P < 0.001). Statistically significant results depending on the number of bins were equal to P = 0.012 (8–256 bins), P = 0.010 (32–1024 bins), P = 0.009 (16–256 bins), P = 0.001 (32–128 bins), and P < 0.001 .Figure When an absolute discretization was adopted, all normalization methods improved the number of robust features compared to no normalization, irrespective of the MR sequence , 0.76 , and 0.78  when the Nyul, WhiteStripe and Z-Score methods were applied, respectively. Absolute improvement was poor for the T2w-flair sequence and did not exceed 0.04 for comparisons of no normalization and the Z-Score method  and T2w-flair (P = 0.597) sequences.The use of an MR intensity normalization method significantly improved the balanced accuracy in DATASET2 for the T1w-gd sequence  for which intensities have a physical meaning. In MRI, intensity values strongly depend on acquisition parameters, making the generalization of radiomics models even more challenging38. Recently, the IBSI has proposed recommendations for each imaging modality43. For MRI, a relative discretization is recommended to account for the variable intensity ranges.Radiomics relies on the extraction of features from multimodal imaging, aiming to improve patient care. Although acquisition parameters strongly affect the content of MR images, only some recent studies have specifically focused on the impact of MRI pre-processing methods on radiomics features33. Additionally, piecewise mapping can be distorted when large tumours are considered. These observations are in accordance with our results showing that different texture feature values were obtained with the Nyul method compared to no normalization and the WhiteStripe and Z-Score methods with FBN discretization . Three types of intensity binning were compared: (1) a constant bin size and relative bounds (FBS); (2) a constant number of bins and relative bounds (FBN); and (3) a constant number of bins and absolute bounds. For 20 out of the 240 indices, patient rankings obtained with binning (1) and (2) were highly correlated (|r|> 0.7). This number increased to 188 when comparing rankings obtained with binning (2) and (3) and was reduced to 9 when comparing (2) and (3). They subsequently adopted the absolute discretization (1), as it does not require the setting of absolute lower and upper bounds. Goya-Outi et al. have shown similar patient rankings for the large majority of 240 textural features when using different values of FBN  or FBS . More recently, Duron et al. evaluated the influence of grey-level discretization on inter- and intra-observer reproducibilities of textural features extracted from 6 MR sequences39 based on manual and automatic segmentations. FBS was shown to be associated with a higher number of reproducible features based on a combination of ICCs and CCCs. In this study, the authors did not normalize the intensities before feature extraction, but they also did not limit the conclusions to a selected range of bin sizes or numbers. In our study, we found that the choice of the number of bins leads to small differences between 16 and 128 bins, with a maximum variation of 10% in the percentages of robust features  extracted from different glioblastoma (GBM) subregions . They showed that radiomics features extracted from necrotic regions were the most reproducible and recommended that, after the bias field correction step, noise filtering should be applied. In that work, no analysis of the optimal pre-processing based on a clinical classification or regression task was performed, making it difficult to compare their results to ours. In 2019, Um et al.40 studied the impact of image pre-processing methods on 420 radiomics features extracted from MR images from two datasets: 50 patients from the TCGA-GBM dataset and 111 institutional patients. They evaluated five image pre-processing techniques: 8-bit global rescaling, 8-bit local rescaling, bias field correction, histogram normalization and isotropic resampling. Their goal was to evaluate the ability of a machine learning classifier to classify each patient according the cohort to which a patient belongs (covariate shift) depending on the pre-processing step performed. They also assessed the impact of each pre-processing step on an overall survival model. They showed that no single pre-processing step was sufficient to completely remove the machine effect. However, in their cohort, histogram normalization combined with a relative grey-level discretization  was the most important step in reducing inter-machine effects. Compared to our study, they did not analyse the impact of different methods of normalization or discretization. Moreover, the comparison of their results to ours is difficult, as no interplay effect of the different pre-processing methods was analysed. In addition, there was no use of "skull stripping" prior to the application of intensity rescaling, which should have been a mandatory step47. Finally, this comparison is also challenging due to different cohorts and tasks applied.Most recently, 2 publications focused on the image pre-processing steps and their impact on radiomics feature reproducibility in brain patients. Moradmand et al.50. Recently, a compensation method to pool radiomics features from different centres has been suggested. This data-driven post-processing method, called ComBat51, seems to be able to harmonize radiomics data a posteriori. Initially proposed to correct batch effects in genomic studies, ComBat has demonstrated its effectiveness in PET52 and CT53. The next step will consist of comparing ComBat with the pre-processing methods described in this article.Additional studies are awaited to confirm our results, which also need to be validated in other tasks. Of note, cross-validation was used to assess classification performances. Even if the use of an independent test set would have been preferable, the various train-test partitions combined with a bootstrapping strategy allowed us to draw conclusions efficiently. Regularization methods will have to be implemented in future studies to decrease the risk of overfitting. In addition, only anatomical MR sequences have been considered. These images are, however, the conventional sequences for radiological assessment of cerebral lesions; the use of more quantitative functional imaging is still sparse in clinical practice. In this study, a unique ROI was delineated; thus, the choice of the ideal number of bins can be influenced by the sharpness of the intensities at the border of the lesion. As the number of voxels included in the tumour was negligible compared to the number of voxels in the whole brain , no tumour exclusion was applied during the normalization process. This assumption could have biased, to a limited extent, the implementation of the normalization algorithms. In the second experiment, in which a classification task was studied, the results from DATASET1 regarding feature reproducibility were considered for feature selection in model 4. In DATASET1, a narrow set of acquisition and reconstruction parameters was investigated and compared to real-life disparity, emphasizing the need for additional studies. Finally, some pre-processing step parameters, such as bias field correction and spatial resampling, could have affected comparisons. These two pre-processing methods have still been used in a large number of published studies that have demonstrated their importance for the robustness of featuresIn conclusion, a standardized pre-processing pipeline is recommended for brain tumour radiomics analyses. For models based on first- and second-order features, the combination of Z-Score normalization and absolute discretization seems to be the best of the methods tested. For works that consider only second-order features, the relative discretization without prior intensity normalization seems to be sufficient. Even if the bin number for the discretization has a small impact on classification performances, 32 bins appear to be a good compromise when T1w-gd and T2w-flair sequences are considered. The pre-processing methods used must be described in detail in the published papers to achieve reliable radiomics-based machine learning models. Such a pipeline will be pivotal for the implementation of large-scale multicentric studies and may pave the way for the development and validation of MR-based radiomics biomarkers.54. In this manuscript, the same cohort was considered to evaluate the stability of first-order and second-order radiomics features across acquisitions. Each patient underwent two MR acquisitions on 1.5 T  and 3 T  scanners, with a mean interval of 7.4 (± 3.0) days. Inclusion criteria supposed that no clinical or morphological change related to the glioma occurred during this delay. This was certified by a blinded radiologist . A post-contrast 3D axial T1-weighted (T1w-gd) sequence and an axial T2-weighted fluid attenuation inversion recovery (T2w-flair) sequence were acquired on each scanner.Two retrospective datasets were used for this study. DATASET1 included twenty consecutive patients with WHO grade II and III gliomas between January and June 2010 Table . A previ57.DATASET2 included pre-operative multi-institutional scans of The Cancer Genome Atlas (TCGA) Glioblastoma Multiforme (GBM) and Low-Grade Glioma (LGG) collections, publicly available in The Cancer Imaging Archive (TCIA). A total of 135 and 108 exams, including T1w-gd and T2w-flair sequences extracted from the TCGA-GBM and TCGA-LGG cohorts, respectively, were used 58 with default parameters. They were then spatially resampled on a 1 mm × 1 mm × 1 mm grid as suggested by Vallières et al.50 using b-spline interpolation with ANTs. Images from DATASET1 were finally skull-stripped with the Brain Extraction Tool (BET) of the FSL software (FMRIB's Software Library)59 and co-registered with a global linear registration including 12 degrees of freedom using ANTs to the T1w-gd sequence, considered as the reference. As some differences occurred in the skull stripping between the 1.5 T and 3 T images for the same MR sequence, an intersection between the two masks was performed. For DATASET2, the method described by Bakas et al. was used for co-registration to recover the spatial domain in which the segmentations were performed60. Brain masks provided by Bakas et al. were applied for skull stripping. In both cases, MR images were finally normalized using 3 different methods .MR images from DATASET1 and DATASET2 were first corrected for the bias field effect using the N4ITK algorithmThe Z-Score method normalizes image histograms by subtracting 33. As conventionally applied in the literature, the “white stripe” region was defined automatically in this work, using a threshold in intensities, corresponding to ± 5% of The WhiteStripe method normalizes image intensities by subtracting 31. The normalization problem is addressed by learning a standard histogram from a set of images and linearly mapping the intensities of each image of interest to this standard histogram. The standard histogram is learned by averaging predefined landmarks deduced from histograms of the training set. The intensity landmark configuration 32.Nyul’s method corresponds to piecewise linear histogram matchingNote that for the normalization process, no tumour exclusion from the brain mask was applied.33. The code used in this paper as well as details about the algorithm implementation61 are available at https://github.com/jcreinhold/intensity-normalization.More details about intensity normalization methods can be found in the original papershttps://www.slicer.org). For DATASET2, the labelled regions supplied by Bakas et al. were merged.A unique ROI including the tumour and peritumoral oedema was considered. These ROIs were segmented for DATASET1 by an experienced radiation oncologist  using the 3D Slicer open-source platform version 4.10.1 , grey-level run length matrix , grey-level size zone matrix , neighbourhood grey tone difference matrix  and grey-level dependence matrix . Except for 4, all the features conformed to the definition provided by the Imaging Biomarker Normalization Initiative (IBSI)43. All the features used in this study are listed in Supplementary Data The open-source Pyradiomics package (version 2.1.2) was used to extract 18 first-order statistics and 73 textural features from the segmented tumour regions of both DATASETSTo assess the impact of the intensity discretization method on textural features, two approaches of grey-level discretization commonly used in the literature were implemented.The FBS method assigns the same bin for every voxel intensity corresponding to the bin width The FBN method discretizes every voxel intensity from an ROI to a fixed number of To correctly analyse the impact of grey-level discretization on pre-processed images on which the intensity ranges can be different, a scaling factor was computed for the FBS method, as shown in Eq. :5\docume63. A one-way analysis of variance (ANOVA) test was conducted to compare JSD values among the normalization methods. If the ANOVA test was statistically significant, a subsequent pairwise post hoc Tukey’s multiple comparison test was performed. For both tests, a P value < 0.05 was considered significant. The CCCs and ICCs were computed to assess the stability of first-order and textural features across the two acquisitions before and after normalization  was used for the statistical analysis. Regarding DATASET1, JSD was used to compare each pair of intensity histograms before and after normalization65. These included random forest, naïve Bayes, logistic regression, support vector machine and neural network multi-layer perception classifiers. Default parameters were chosen to prevent overfitting. Multiple classifiers were used to avoid limiting the conclusions to a single machine learning model. Moreover, a five-fold stratified cross-validation was adopted. In all cases, feature values were normalized using the Z-Score method within the cross-validation. The average values of the balanced accuracies and the ROC-AUC and corresponding 95% CIs evaluated using the five left-out folds of the 5 machine learning models were reported. For the 95% CIs, bootstrapping including 1000 iterations was applied. Balanced accuracy is a performance metric that should be preferred to accuracy in the case of imbalanced datasets66. Model 1 included first-order features alone. Model 2 was based on textural features only. The added value of the combination of the two types of features was analysed in model 3. Model 4 included only features defined as robust, i.e., having both an ICC and a CCC > 0.8 in the DATASET1 experiment. A two-way ANOVA test was conducted to simultaneously evaluate the effect of normalization and discretization. If the ANOVA test was statistically significant, a subsequent pairwise post hoc Tukey’s multiple comparison test was performed. For both tests, a P value < 0.05 was considered significant.DATASET2 aimed to evaluate the usefulness of intensity normalization and to define the optimal grey-level discretization for a tumour grade classification task. Five widely used classifiers were implemented based on the scikit-learn library version 0.20.3The design of the study is detailed in Fig. Supplementary information.